Fig. 3

UTMD inhibited autophagic flux in IR-treated glioblastoma cells. a, b Representative immunoblot and quantification analysis of p62 after GL261 and U251 cells were treated with UTMD-L (ultrasonic intensity of 1.2 W/cm² combined with 100 μl/ml MBs) or UTMD-H (ultrasonic intensity of 1.2 W/cm² combined with 200 μl/ml MBs) at a duty cycle of 10% for 60 s followed by IR (2 Gy) exposure. ACTB was used as an internal standard for protein loading. c, d GL261 and U251 cells were pretreated with RAPA (20 nmol/L) or BafA1 (10 nmol/L) for 1 h and then cells were treated with UTMD (ultrasonic intensity of 1.2 W/cm² combined with 200 μl/ml MBs at a duty cycle of 10% for 60 s) followed by IR (2 Gy) exposure. Total cell lysates were immunoblotted with anti-p62, anti-LC3B, and anti-ACTB antibodies and the bar graphs show the quantification of LC3B2 and p62. e–h GL261 and U251 cells were transfected with lentiviral vectors encoding ATG5 or ATG5 siRNA. Then, the cells were treated with UTMD (ultrasonic intensity of 1.2 W/cm² combined with 200 μl/ml MBs at a duty cycle of 10% for 60 s) followed by IR (2 Gy) exposure. The expression of LC3B2, p62 and ATG5 was detected by Western blotting. The bar graphs show the quantification of LC3B2, p62 and ATG5. i, j GL261 and U251 cells were pretreated with 3-MA (5 mmol/L) for 1 h and then exposed to UTMD (ultrasonic intensity of 1.2 W/cm² combined with 200 μl/ml MBs at a duty cycle of 10% for 60 s) followed by IR (2 Gy) exposure. Total cell lysates were immunoblotted with anti-p62, anti-LC3B, and anti-ACTB antibodies. The bar graphs show the quantification of LC3B2 and p62. Values are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. the vehicle-treated control group; ^#P < 0.05, ^##P < 0.01 vs. IR group; ^&P < 0.05, ^&&P < 0.05 vs. IR plus UTMD group. IR ionizing radiation, UTMD ultrasound-triggered microbubble destruction, ACTB β-actin, LC3B light chain 3 beta, p62 sequestosome 1, RAPA rapamycin, BafA1 bafilomycin A1, 3-MA 3-methyladenine, GFP green fluorescent protein, MBs microbubbles, SD standard deviation, a.u. arbitrary units